蛋白質在生物體內的位置、轉移與交互作用等，往往提供生物學研究重要資訊。對蛋白質標記螢光分子具有快速、靈敏度高、專一性結合且方便觀察的特點，是廣泛應用於生物體內的方法之一。
本論文選擇以SNAP-tag蛋白標記技術，利用SNAP-tag蛋白對O6-BG基團的專一辨識性且以共價鍵的方式鍵結，發展兩種類型螢光探針: (一)籠閉型苄基鳥嘌呤螢光探針 ；(二)尼羅染料螢光探針。第一類型探針在鳥嘌呤基團N-9位置修飾上辨識端，形成籠閉型式探針，期望藉由加入目標分析物切斷辨識端後，使裸露的鳥嘌呤基團會與SNAP-tag蛋白結合，而有螢光增益的現象，達到偵測目標分析物的。第二類型計畫是開發近紅外光尼羅染料螢光探針，近紅外螢光具有低生物背景訊號干擾、低光損傷害以及較好的生物組織穿透性等，利用對極性敏感螢光探針的特性，成功地開發了SNAP-tag蛋白標記之螢光探針，並應用在細胞標記影像中。

Protein functions and dynamics in living cells can provide important information in biological research and medical diagnosis. The uses of fluorescent probes to study proteins are advantageous, as they allow for fast, high sensitivity, high specificity and ease for observation. In this thesis, I have designed two novel fluorescent probes for SNAP-tag which is an important protein in chemical biology for specific and covalent reaction with probes consisting O6-benzylguanine moiety. The first probe is based on the caged-benzylguanine concept by chemical modification of benzylguanine N-9 position to prevent the reaction of benzylguanine and SNAP-tag. Upon uncaging with the target molecule, the fluorescent dye can then be transferred to the SNAP-tag or MGMT (benzylguanine wild type protein) to emit stronger fluorescence. In the second fluorescent probe, we have successfully developed two near-infrared fluorescent probes based on Nile red and Nile blue, respectively, for fluorescent turn-on labeling of SNAP-tag protein. We have successfully applied these two probes for no-was and highly organelle specific labeling of SNAP-tag proteins in living cells.

摘要 i
Abstract ii
謝誌 iii
目錄 iv
第一章、緒論 1
§1-1 酶蛋白及非酶蛋白質(protease and non-enzymatic protein) 1
§1-2 蛋白質標記(protein labeling) 3
§1-2-1 螢光蛋白標記 4
§1-2-2 螢光分子標記 6
第二章、文獻回顧 13
§2-1螢光共振能轉量移螢光探針(Förster Resonance Energy Transfer, FRET) 13
§2-2自組裝式螢光探針(self-assembling fluorescent probes) 15
§2-3籠閉式螢光探針(caged fluorescence probes) 16
§2-4環境敏感螢光探針(environment-sensitive fluorophore) 18
第三章、探針之設計構想 24
§3-1 SNAP-tag 標記技術 24
§3-2 探針設計與策略 26
§3-2-1 籠閉型苄基鳥嘌呤螢光探針 26
§3-2-2 尼羅染料螢光探針 29
第四章、實驗結果與討論 32
§4-1 籠閉型苄基鳥嘌呤螢光探針測試 32
§4-2 尼羅染料(Nile dyes) 40
§4-2-1尼羅紅染料螢光探針測試 (Nile red) 40
§4-2-2尼羅藍染料螢光探針測試 (Nile blue) 48
第五章、實驗結論 54
第六章、實驗部分 55
§6-1 一般實驗敘述 55
§6-2 有機合成及光譜資料 57
參考文獻 74
附錄 81

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